In this proposal we lay out a comprehensive research plan to evaluate our vaccination strategies targeting Clostridium difficile to prevent or treat infections. With the emergence of highly antibiotic-resistant bacterial strains, new approaches for combating bacterial infections are desperately needed. In this research proposal, Sorrento Therapeutics Inc. (STI) together with researchers from the University of Iowa have outlined experiments that will harness a powerful combination of chemistry, microbiology, and immunology to provide a solid rational basis for the development and evaluation of a quorum quenching vaccine to prevent C. difficile infections. We will first evaluate passive vaccination targeting the agr quorum sensing system of C. difficile for the protection against infections caused by antibiotic-sensitive and -resistant Clostridium difficile strains. We will then establish active vaccination protocols that result in high quorum quenching antibody titers as the basis for our ultimate goal, the development of an effective C. difficile vaccine. The specific projects of our STTR research proposal are (1) Isolate neutralizing human monoclonal anti- AIP antibodies. To establish the therapeutic efficacy of passive anti-AIP immunotherapy we will isolate anti- AIP-1 and AIP-2 binders from our proprietary human monoclonal antibody phage display library, convert them to IgGs, produce and purify them, and use an assay created by our collaborator, Dr. Horswill (University of Iowa), to select the best candidates for testing in an in vivo hamster challenge model. (2) Demonstrate protection by selected human anti-CDAP mAbs in a C. difficile hamster infection model in vivo. STI will produce four fully human QQ CDAP antibodies for in vivo evaluation in the standard C. difficile hamster infection model, to be performed by Dr. Ellermeier (University of Iowa). (3) Demonstrate protection by an anti-AIP active vaccine in a C. difficile hamster infection model in vivo. Accomplishment of projects (1) and (2) will prove the concept that interference with quorum sensing in vivo provides protection from C. difficile infection. In this final project, the same CDAP immunogens that were used to isolate the protective mAbs from the STI human mAb phage display library will be used to actively immunize hamsters prior to C. difficile challenge. Given our preliminary data and the expertise of the investigators, we believe there is a high probability of success for this project. In a subsequent Phase II application we would test carriers and adjuvants in combinations in the C. difficile hamster infection model and a neo-natal pig model. Antibody titers will be determined prior to C. difficile challenge and the best vaccine candidate developed. The proposed product, a unique C. difficile vaccine that targets quorum sensing, would provide a much needed alternative to antibiotic management of a serious and escalating health threat.